Method for inducing alveolar epithelial progenitor cells

ABSTRACT

This invention provides a method for stably producing alveolar epithelial progenitor cells from pluripotent stem cells, including steps of culturing pluripotent stem cells in (1) a medium containing activin A and a GSK3β inhibitor, (2) a medium containing a BMP inhibitor and a TGFβ inhibitor, and (3) a medium containing BMP4, retinoic acid, and a GSK3β inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.14/783,382, filed Oct. 8, 2015, which is a 371 of PCT/JP2014/061106,filed Apr. 14, 2014, which in turn claims the benefit of Japanese PatentApplication No. 2013-084034, filed Apr. 12, 2013, the contents of eachof which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a method for producing alveolarepithelial progenitor cells from pluripotent stem cells. The presentinvention also relates to a kit used for producing alveolar epithelialprogenitor cells from pluripotent stem cells.

BACKGROUND ART

The lung is one of the most complicated organs, and it is considered tobe composed of approximately 40 different types of cells. Among them,the pulmonary alveolus is composed of the alveolar space, which storesgas, and the alveolar epithelium, which surrounds the same. In addition,the alveolar epithelium is composed of the type I alveolar epithelialcells and the type II alveolar epithelial cells. The former forms ablood-air barrier with the microvascular endothelium surrounding thepulmonary alveolus with the aid of the basal membrane and exchanges theintra-alveolar gas with the blood gas. The latter comprises manylamellar corpuscles, it undergoes exocytosis of pulmonary surfactants,and it forms the alveolar lining layer.

In recent years, cells having pluripotency, such as embryonic stem cells(ES cells) or induced pluripotent stem cells (iPS cells) obtained byintroducing undifferentiated-cell-specific genes into somatic cells,have been reported (U.S. Pat. No. 5,843,780 and WO 2007/069666), methodsfor inducing alveolar epithelial cells from such cells have beenreported (Rippon H. J. et al, Cloning Stem Cells 6: 49-56, 2004; CorauxC. et al, Am. J. Respir. Cell Mol. Biol., 32:87-92, 2005; and MorriseyE. E and Hogan B. L., Dev. Cell., 18: 8-23, 2010), and growth factorsand the like that are necessary for the induction of such cells havealso been reported. However, there are no examples demonstrating theefficient induction of human pulmonary alveolar cells.

Examples of diseases that destroy the pulmonary alveolus includeemphysema, interstitial pneumonia, and lymphangioleiomyomatosis. Inparticular, emphysema is treated via symptomatic treatment orconservative treatment at present, and there is no radical treatmenttherefor. In addition, there is no radical treatment available for otherpulmonary alveolar diseases, and the development of cell-transfertreatment has been accordingly awaited.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method forproducing alveolar epithelial progenitor cells from pluripotent stemcells. It is another object of the present invention to provide a kitused for producing alveolar epithelial progenitor cells from pluripotentstem cells.

The present inventors have conducted concentrated studies in order toattain the above objects. As a result, they discovered that pluripotentstem cells could be induced to differentiate into alveolar epithelialprogenitor cells with the use of various growth factors and compounds.This has led to the completion of the present invention.

Specifically, the present invention includes the following.

-   [1] A method for producing alveolar epithelial progenitor cells from    pluripotent stem cells comprising Steps (1) to (3) below:

(1) culturing pluripotent stem cells in a medium containing activin Aand a GSK3β inhibitor;

(2) culturing the cells obtained in Step (1) in a medium containing abone morphogenic protein (BMP) inhibitor and a TGFβ inhibitor; and

(3) culturing the cells obtained in Step (2) in a medium containingBMP4, retinoic acid, and a GSK3β inhibitor.

-   [2] The method according to [1], which further comprises a step of    extracting CPM-positive cells as alveolar epithelial progenitor    cells, following Step (3).-   [3] The method according to [1] or [2], wherein Step (1) further    comprises culturing pluripotent stem cells with the addition of an    ROCK inhibitor and/or HDAC inhibitor to a medium.-   [4] The method according to any one of [1] to [3], wherein Step (1)    comprises culturing for 6 days or longer.-   [5] The method according to any one of [1] to [4], wherein Step (2)    comprises culturing for 4 days or longer.-   [6] The method according to any one of [1] to [5], wherein Step (3)    comprises culturing for 4 days or longer.-   [7] The method according to any one of [1] to [6], wherein the GSK3β    inhibitor is CHIR99021, the BMP inhibitor is Noggin, and the TGFβ    inhibitor is SB431542.-   [8] The method according to any one of [3] to [7], wherein the ROCK    inhibitor is Y-27632 and/or the HDAC inhibitor is sodium butyrate.-   [9] The method according to any one of [1] to [8], which further    comprises Steps (4) and (5), following Step (3) below:

(4) culturing the cells obtained in Step (3) in a medium containingFGF10; and

(5) culturing the cells obtained in Step (4) in a medium containing asteroid drug, a cAMP derivative, a phosphodiesterase inhibitor, and KGF.

-   [10] The method according to [9], which further comprises a step of    extracting CPM-positive cells as alveolar epithelial progenitor    cells, following Step (5).-   [11] The method according to [9] or [10], wherein Step (4) comprises    culturing for 7 days or longer.-   [12] The method according to any one of [9] to [11], wherein    Step (5) comprises culturing for 4 days or longer.-   [13] The method according to any one of [9] to [12], wherein the    steroid drug is dexamethasone, the cAMP derivative is 8Br-cAMP, and    the phosphodiesterase inhibitor is 3-isobutyl-1-methylxanthine    (IBMX).-   [14] The method according to any one of [1] to [13], wherein the    alveolar epithelial progenitor cells are human alveolar epithelial    progenitor cells.-   [15] A method for producing alveolar epithelial progenitor cells,    which further comprises a step of three-dimensional culture of the    alveolar epithelial progenitor cells produced by the method    according to [1] or [2].-   [16] Alveolar epithelial progenitor cells produced by the method    according to any one of [1] to [15].-   [17] A kit used for producing alveolar epithelial progenitor cells    from pluripotent stem cells, which comprises activin A, a GSK3β    inhibitor, a BMP inhibitor, a TGFβ inhibitor, BMP4, retinoic acid, a    steroid drug, a cAMP derivative, a phosphodiesterase inhibitor,    FGF10, and KGF.-   [18] The kit according to [17], which further comprises an ROCK    inhibitor and/or HDAC inhibitor.-   [19] The kit according to [17] or [18], wherein the GSK3β inhibitor    is CHIR99021, the BMP inhibitor is Noggin, the TGFβ inhibitor is    SB431542, the steroid drug is dexamethasone, the cAMP derivative is    8Br-cAMP, and the phosphodiesterase inhibitor is IBMX.-   [20] The kit according to [18] or [19], wherein the ROCK inhibitor    is Y-27632 and/or the HDAC inhibitor is sodium butyrate.-   [21] A method for extracting alveolar epithelial progenitor cells,    which comprises a step of extracting CPM-positive cells as alveolar    epithelial progenitor cells from a cell population including    alveolar epithelial progenitor cells.

This description includes part or all of the content as disclosed in thedescription and/or drawings of Japanese Patent Application No.2013-084034, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B each show a scheme for producing alveolar epithelialprogenitor cells from pluripotent stem cells. In the figures, “Stage I”is synonymous with “Step 1,” “Stage II” is synonymous with “Step 2,”“Stage III” is synonymous with “Step 3,” “Stage IV” is synonymous with“Step 4,” and “Stage V” is synonymous with “Step 5.” Hereafter, such“stages” and “steps” are collectively referred to as “Steps.”

FIG. 2A shows images of immunostained CPM and NKX2-1 in the cells afterthe completion of Step 3. FIG. 2B shows images of immunostained CPM andNKX2-1 in the cells after the completion of Step 2 (upper images) andStep 3 (lower images).

FIG. 3A shows percentages of CPM-positive cells after sorting viaCPM-based MACS, after the completion of Step 3. In the figure, “Isotypecontrol” shows the results for the negative control, “Pre-sorting” showsthe results for the cells before sorting via MACS, “CPM positiveselection” shows the results for CPM-positive cells after sorting viaMACS, and “CPM negative selection” shows the results for CPM-negativecells after sorting via MACS. FIG. 3B shows percentages of CPM-positivecells after sorting via CPM-based MACS after the completion of Step 3.“Pre-sorting” shows the results for the cells before sorting via MACS,“CPM(+) sorting” shows the results for the MACS-sorted CPM-positivecells, and “CPM(−) sorting” shows the results for the MACS-sortedCPM-negative cells.

FIG. 4A shows images of immunostained CPM and NKX2-1 in the MACS-sortedCPM-positive cells (CPM(+) sorting) and in the CPM-negative cells(CPM(−) sorting) after the completion of Step 3. FIG. 4B shows images ofimmunostained NKX2-1 in the MACS-sorted CPM-positive cells (CPM(+)sorting) and in the CPM-negative cells (CPM(−) sorting) after thecompletion of Step 3.

FIG. 5A shows percentages of NKX2-1-positive cells among the MACS-sortedcells after the completion of Step 3. In the figure, “Pre-sorting” showsthe results for the cells before sorting via MACS, “CPM positiveselection” shows the results for the MACS-sorted CPM-positive cells, and“CPM negative selection” shows the results for the MACS-sortedCPM-negative cells. FIG. 5B shows percentages of NKX2-1-positive cellsamong the MACS-sorted cells after the completion of Step 3. In thefigure, “CPM⁺ sorted cells” shows percentages of NKX2-1-positive cellsamong the MACS-sorted CPM-positive cells (92.3±0.7%), and “CPM⁻ sortedcells” shows percentages of NKX2-1-positive cells among the MACS-sortedCPM-negative cells (22.2±2.3%).

FIG. 6 shows the assay results of CPM mRNA levels (left) and NKX2-1 mRNAlevels (right) in the MACS-sorted cells on the basis of CPM markers viaquantitative PCR after the completion of Step 3. “Step 3 ” shows theresults for the cells before sorting, “Step 3 (+)” shows the results forthe CPM-positive cells, “Step 3 (−)” shows the results for theCPM-negative cells, “Fetal Lung” shows the results for the fetalpneumocytes, and “Adult Lung” shows the results for the adultpneumocytes.

FIG. 7 shows the assay results of CPM mRNA levels (left) and NKX2-1 mRNAlevels (right) in the MACS-sorted cells via quantitative PCR after thecompletion of Step 4. “Step 4” shows the results for the cells beforesorting, “Step 4 (+)” shows the results for the CPM-positive cells,“Step 4 (−)” shows the results for the CPM-negative cells, “Fetal Lung”shows the results for the fetal pneumocytes, and “Adult Lung” shows theresults for the adult pneumocytes.

FIG. 8A shows images of immunostained iPS cells (201B7) in theMACS-sorted CPM-positive cells after the completion of Step 5. FIG. 8Bshows images of immunostained iPS cells (SFTPC reporter cells;SFTPC-reporter 201B7) in the MACS-sorted CPM-positive cells after thecompletion of Step 5. FIG. 8C shows percentages of EGFP-positive iPScells (SFTPC reporter cells; SFTPC-reporter 201B7) among the MACS-sortedCPM-positive cells after the completion of Step 5.

FIG. 9A and FIG. 9B each show an image of immunostained cells after thecompletion of Step 5.

FIG. 10 shows the assay results of mRNA levels via quantitative PCR ofCPM, NKX2-1, SFTPA2, SFTPB, SFTPC, DCLAMP, CCSP (SCGB1A1), SCGB3A2, andNGFR in the MACS-sorted cells after the completion of Step 5. “Step 5”shows the results for the cells before sorting, “Step 5 (+)” shows theresults for the CPM-positive cells, “Step 5 (−)” shows the results forthe CPM-negative cells, “Fetal Lung” shows the results for the fetalpneumocytes, and “Adult Lung” shows the results for the adultpneumocytes.

FIG. 11 shows images of CPM in the human fetal lung tissue subjected tostaining together with NKX2-1, SFTPC, or T1α serving as a marker of theprenatal period, the adenoid period, or the canalicular period of thelung, respectively.

FIG. 12 shows images of CPM in the mouse lung tissue at relevantprenatal periods (E12.5, E15.5, and E17.5) subjected to stainingtogether with NKX2-1 serving as a marker of the prenatal period, theadenoid period, or the canalicular period of the lung, respectively.

FIG. 13 shows a summary of a method of sorting CPM-positive cells fromamong the cells after the completion of Step 3 and culturing the sortedcells together with human fetal pulmonary fibroblasts.

FIG. 14A shows transmission electron microscopic images of spheroidsfollowing the three-dimensional culture of the CPM-positive cells afterthe completion of Step 3. In FIG. 14A, the images at the center and onthe right are each an enlarged view of a part of the image on the left.FIG. 14B shows transmission electron microscopic images of type IIalveolar epithelial cells of mouse lungs. The image on the right showsan enlarged view of a part of the image on the left. FIG. 14C showstransmission electron microscopic images of the mouse fetal lung atE17.5. The image on the right shows an enlarged view of a part of theimage on the left. In the figures, “Lu” indicates a lumen.

FIG. 15A shows hematoxylin-eosin-stained images of thethree-dimensionally cultured CPM-positive cells (left) and CPM-negativecells (right) after the completion of Step 3. The lower images showenlarged views of the upper images. FIG. 15B shows images of thespheroids immunostained with the CPM, NKX2-1, GFP (SFTPC), SFTPC(endogenous), and AQP5 antibodies following the three-dimensionalculture of the CPM-positive cells after the completion of Step 3.

FIG. 16 shows images of the spheroids immunostained with the CPM,NKX2-1, SFTPC, SFTPB, SFTPA, SFTPD, ID2, and SOX9 antibodies followingthe three-dimensional culture of the CPM-positive cells after thecompletion of Step 3. The images on the right show images of alveolarepithelial cell-associated proteins subjected to dual staining witheither CPM or NKX2-1.

FIG. 17 shows images of the spheroids immunostained with the T1α, SFTPC,AQP5, and CAV1 antibodies following the three-dimensional culture of theCPM-positive cells after the completion of Step 3.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereafter, the present invention is described in detail.

The present invention provides a method for producing alveolarepithelial progenitor cells (e.g., human alveolar epithelial progenitorcells) from pluripotent stem cells comprising steps of culturingpluripotent stem cells in (1) a medium containing activin A and a GSK3βinhibitor, (2) a medium containing a BMP inhibitor and a TGFβ inhibitor,and (3) a medium containing BMP4, retinoic acid, and a GSK3β inhibitor.

The method for producing alveolar epithelial progenitor cells accordingto the present invention may comprise a step of extracting CPM-positivecells as the alveolar epithelial progenitor cells, following Step (3).

The method for producing alveolar epithelial progenitor cells accordingto the present invention may further comprise Step (4) of culture in amedium containing FGF10 and Step (5) of culture in a medium containing asteroid drug, a cAMP derivative, a phosphodiesterase inhibitor, and KGF,following Step (3). In addition, the method may further comprise a stepof extracting CPM-positive cells as the alveolar epithelial progenitorcells, following Step (5).

In the present invention, the term “alveolar epithelial progenitorcells” refers to progenitor cells of type I alveolar epithelial cells ortype II alveolar epithelial cells, which express CPM or NKX2-1. In thisdescription, the term “alveolar epithelial cells” is not distinguishedfrom the term “alveolar epithelial progenitor cells,” unless otherwisespecified. In the present invention, “CPM” indicates a polynucleotideshown in the NCBI Accession Number NM_001005502, NM_001874, or NM_198320or a protein encoded thereby. In the present invention, “NKX2-1 ”indicates a polynucleotide shown in the NCBI Accession NumberNM_001079668 or NM_003317 or a protein encoded thereby.

In the present invention, examples of markers for alveolar epithelialprogenitor cells include polynucleotides selected from the groupconsisting of SFTPB (NCBI Accession Numbers NM_000542 and NM_198843),SFTPC (NCBI Accession Numbers NM_001172357, NM_001172410, andNM_003018), and CCSP (NCBI Accession Number NM_003357) and proteinsencoded by such polynucleotides.

[Step of Culture in a Medium Containing Activin A and a GSK3β Onhibitor]

A medium used in the step of culturing pluripotent stem cells accordingto the present invention can be prepared from a medium used for animalcell culture as a basal medium. Examples of basal media include IMDMmedium, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEMmedium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium,RPMI 1640 medium, Fischer's medium, Neurobasal Medium (LifeTechnologies), and a mixture of any such media. A medium may or may notcontain blood serum. A medium may optionally contain one or more serumsubstitutes selected from among, for example, albumin, transferrin,Knockout Serum Replacement (KSR) (an FBS serum substitute used for EScell culture), N2 supplements (Invitrogen), B27 supplements(Invitrogen), fatty acid, insulin, collagen precursors, trace elements,2-mercaptoethanol, and 3′-thiol glycerol. In addition, a medium cancontain one or more substances selected from among, for example, lipids,amino acids, L-glutamine, Glutamax (Invitrogen), nonessential aminoacids, vitamins, growth factors, low-molecular-weight compounds,antibiotics, antioxidants, pyruvic acids, buffer agents, and inorganicsalts. RPMI 1640 medium supplemented with B27 and antibiotics ispreferable.

In this step, pluripotent stem cells are cultured in a medium preparedby supplementing the basal medium described above with activin A and aGSK3β inhibitor. In this step, an HDAC inhibitor may further be added.

Activin A is a homodimer with two beta A chains, the amino acid sequenceof activin A is 100% homologous to that of a protein of a human, mouse,rat, pig, cow, or cat, and, accordingly, the relevant species are notparticularly limited. In the present invention, activin A is preferablyof an active form with the N-terminal peptide being cleaved, and it ispreferably a homodimer comprising, bound thereto via a disulfide bond,the Gly311-Ser426 fragment with the N-terminal peptide of the inhibinbeta A chain (e.g., NCBI Accession Number NP_002183) being cleaved. Suchactivin A is commercially available from, for example, Wako and R&DSystems.

The activin A concentration in the medium is, for example, 10 ng/ml, 20ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90ng/ml, 100 ng/ml, 150 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml,600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml, or 1 mg/ml, although theconcentration is not limited thereto. The concentration is preferably100 ng/ml.

The term “GSK362 inhibitor” used herein is defined as a substance thatinhibits kinase activity of the GSK-3β protein (e.g., the capacity forphosphorylation of (β-catenin), and many such substances are alreadyknown. Examples thereof include: an indirubin derivative, such as BIO,which is also known as a GSK-3β inhibitor IX(6-bromoindirubin-3′-oxime); a maleimide derivative, such as SB216763(3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indo1-3-yl)-1H-pyrrole-2,5-dione);a phenyl α-bromomethylketone compound, such as a GSK-3β inhibitor VII(4-dibromoacetophenone); a cell-membrane-permeable phosphorylatedpeptide, such as L803-mts, which is also known as a GSK-3β peptideinhibitor (i.e., Myr-N-GKEAPPAPPQSpP-NH₂); and CHIR99021, such as6-[2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino]ethylamino]pyridine-3-carbonitrile, with high selectivity. While such compoundsare commercially available from, for example, Calbiochem or Biomol, andeasily used, such compounds may be obtained from other companies, orpersons may prepare such compounds by themselves.

A GSK-3β inhibitor that can be preferably used in the present inventionis CHIR99021. In this step, the concentration of CHIR99021 in a mediumis, for example, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 1.5μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, or 50 μM, although theconcentration is not limited thereto. In this step, the concentration ispreferably 1 μM.

The term “HDAC inhibitor” is defined as a substance that inhibits orinactivates enzyme activity of histone deacetylase (HDAC). Examplesthereof include low-molecular-weight inhibitors, such as valproic acid(VPA) (Nat. Biotechnol., 26 (7): 795-797, 2008), trichostatin A, sodiumbutyrate (NaB), MC 1293, and M344; nucleic acid-based expressioninhibitors such as siRNAs and shRNAs against HDAC (e.g., HDAC1 siRNASmartpool □ (Millipore) and HuSH 29mer shRNA Constructs against HDAC1(OriGene)); and DNA methyltransferase inhibitors (e.g., 5′-azacytidine)(Nat. Biotechnol., 26 (7): 795-797, 2008).

An HDAC inhibitor that can be preferably used in the present inventionis sodium butyrate (NaB). The concentration of sodium butyrate (NaB) ina medium is, for example, 1 μM, 10 μM, 50 μM, 100 μM, 250 μM, 500 μM,750 μM, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM, although the concentration isnot limited thereto. The concentration is preferably 250 μM.

In this step, culture may be conducted in a culture vessel treated witha coating agent. A coating agent may be a naturally occurring orartificially synthesized extracellular matrix. Examples thereof includeBD Matrigel, collagen, gelatin, laminin, heparan sulfate proteoglycan,entactin, and a combination of any thereof, with Matrigel beingpreferable.

This step may comprise a process of pluripotent stem cell detachment.Examples of methods for cell detachment include a method of mechanicaldetachment and a method of cell detachment involving the use of a celldetachment solution having protease activity and collagenase activity(e.g., Accutase™ and Accumax™ or a cell detachment solution havingcollagenase activity alone. It is preferable that human pluripotent stemcells be detached with the use of a cell detachment solution havingprotease activity and collagenase activity (and the use of Accutase™ isparticularly preferable).

When the step comprises a process of cell detachment, an ROCK inhibitormay be added to a medium, so as to inhibit pluripotent stem cell deathcaused by detachment.

An ROCK inhibitor is not particularly limited, provided that it caninhibit functions of Rho kinase (ROCK). Examples thereof include:Y-27632((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamidedihydrochloride) (e.g., Ishizaki et al., Mol. Pharmacol., 57, 976-983,2000; Narumiya et al., Methods Enzymol., 325, 273-284, 2000);Fasudil/HA1077 (e.g., Uenata et al., Nature 389: 990-994, 1997); H-1152(e.g., Sasaki et al., Pharmacol. Ther., 93: 225-232, 2002); Wf-536(e.g., Nakajima et al., Cancer Chemother. Pharmacol., 52 (4): 319-324,2003) and derivatives thereof; antisense nucleic acids against ROCK; RNAinterference-inducible nucleic acids (e.g., siRNA); dominant-negativevariants; and expression vectors thereof. Since otherlow-molecular-weight compounds are known as ROCK inhibitors, suchcompounds and derivatives thereof can also be used in the presentinvention (e.g., U.S. Patent Application Publication Nos. 2005/0209261,2005/0192304, 2004/0014755, 2004/0002508, 2004/0002507, 2003/0125344,and 2003/0087919, WO 2003/062227, WO 2003/059913, WO 2003/062225, WO2002/076976, and WO 2004/039796). In the present invention, one or moretypes of ROCK inhibitors can be used.

An ROCK inhibitor that can be preferably used in the present inventionis Y-27632. The Y-27632 concentration is, for example, 100 nM, 500 nM,750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15μM, 20 μM, 25 μM, 30 μM, 40 μM, or 50 μM, although the concentration isnot limited thereto. The concentration is preferably 10 μM.

Concerning culture conditions, culture is conducted at about 30□C to40□C, and preferably at about 37□0C, although the temperature is notlimited thereto. Culture is conducted under an atmosphere of aircontaining CO₂, and the CO₂ concentration is preferably about 2% to 5%.

The culture period is not particularly limited because long-term culturewould not cause any problems. For example, the culture period may be atleast 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, or 12 days. The culture period is preferably at least 6 days,and it is particularly preferably 6 days. When the ROCK inhibitor isadded, the duration of addition is 1 day or 2 days, with 1 day beingpreferable. When the HDAC inhibitor is further added, the addition isinitiated on the day following the initiation of the step, and cultureis conducted for at least 3 days, 4 days, 5 days, 6 days, 7 days, 8days, 9 days, 10 days, or 11 days. Culture is preferably conducted forat least 5 days, and particularly preferably for 5 days, in the presenceof the HDAC inhibitor.

[Step of Culture in a Medium Containing BMP Inhibitor and TGFβInhibitor]

A medium used in this step can be prepared from a medium used for animalcell culture as a basal medium. Examples of basal media include IMDMmedium, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEMmedium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium,RPMI 1640 medium, Fischer's medium, Neurobasal Medium (LifeTechnologies), and a mixture of any such media. A medium may or may notcontain blood serum. A medium may optionally contain one or more serumsubstitutes selected from among, for example, albumin, transferrin,Knockout Serum Replacement (KSR) (an FBS serum substitute used for EScell culture), N2 supplements (Invitrogen), B27 supplements(Invitrogen), fatty acid, insulin, collagen precursors, trace elements,2-mercaptoethanol, and 3′-thiol glycerol. In addition, a medium cancontain one or more substances selected from among, for example, lipids,amino acids, L-glutamine, Glutamax (Invitrogen), nonessential aminoacids, vitamins, growth factors, low-molecular-weight compounds,antibiotics, antioxidants, pyruvic acids, buffer agents, and inorganicsalts. A medium mixture of DMEM and Ham's F12 supplemented withGlutamax, B27, N2, 3′-thiol glycerol, and ascorbic acid is preferable.

In this step, the cells obtained in the previous step (i.e., the step ofculture of pluripotent stem cells in a medium containing activin A and aGSK3β inhibitor) are cultured in a medium prepared by supplementing thebasal medium with a BMP inhibitor and a TGFβ inhibitor.

Examples of BMP inhibitors include: protein-based inhibitors, such asChordin, Noggin, and Follistatin; dorsomorphin (i.e.,6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine)and a derivative thereof (P. B. Yu et al., 2007, Circulation, 116:II_60; P. B. Yu et al., 2008, Nat. Chem. Biol., 4:33-41; J. Hao et al.,2008, PLoS ONE, 3 (8): e2904); and LDN-193189 (i.e.,4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline).Dorsomorphin and LDN-193189 are commercially available fromSigma-Aldrich and Stemgent, respectively.

A BMP inhibitor that can be preferably used in the present invention isNoggin. The concentration of Noggin in a medium is not particularlylimited, provided that BMP can be inhibited. For example, suchconcentration is 1 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml, 30ng/ml, 400 ng/ml, 50 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml, 900 ng/ml,1 μg/ml, or 2 μg/ml, although the concentration is not limited thereto.The concentration is preferably 200 ng/ml.

The term “TGFβ inhibitor” used herein refers to a substance thatinhibits signal transmission from the binding of TGFβ to a receptorleading to SMAD. A TGFβ inhibitor is not particularly limited, providedthat such substance inhibits TGFβ from binding to a receptor; i.e., theALK family, or such substance inhibits phosphorylation of SMAD caused bythe ALK family. Examples thereof include Lefty-1 (e.g., NCBI AccessionNos. mouse NM_010094 and human NM_020997), SB431542(4-(4-(benzo[d][1,3]dioxo1-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide),SB202190 (R. K. Lindemann, et al., Mol. Cancer, 2003, 2: 20), SB505124(GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761,LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO2009/146408), and derivatives thereof.

A TGFβ inhibitor that can be preferably used in the present invention isSB431542. The SB431542 concentration in a medium is not particularlylimited, provided that TGFβ is inhibited. For example, suchconcentration can be 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 60μM, 70 μM, 80 μM, 90 μM, 100 μM, 200 μM, 300 μM, 400 μM, or 500 μM,although the concentration is not limited thereto. The concentration ispreferably 10 μM.

In this step, culture may be conducted in a culture vessel treated witha coating agent. Examples of coating agents include BD Matrigel,collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, anda combination of any thereof, with Matrigel being preferable.

This step may be implemented by exchanging the cell culture mediumobtained in the previous step with the medium described above.Alternatively, cells may be detached and reseeded in a culture vessel.When cells are to be detached, particular cells may be selected, and,for example, SOX17- and/or FOXA2-positive cells may be selected and usedin this step. This method is preferably implemented by means of mediaexchange.

When the step comprises a process of cell detachment, an ROCK inhibitormay be added to a medium, so as to inhibit pluripotent stem cell deathcaused by detachment.

Concerning culture conditions, culture is conducted at about 30□C to40□C, and preferably at about 37□C, although the temperature is notlimited thereto. Culture is conducted under an atmosphere of aircontaining CO₂, and the CO₂ concentration is preferably about 2% to 5%.

The culture period is not particularly limited because long-term culturewould not cause any problems. For example, the culture period may be atleast 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.The culture period is preferably 4 days.

[Step of Culture in a Medium Containing BMP4, Retinoic Acid, and GSK3βInhibitor]

A medium used in this step can be prepared from a medium used for animalcell culture as a basal medium. Examples of basal media include IMDMmedium, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEMmedium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium,RPMI 1640 medium, Fischer's medium, Neurobasal Medium (LifeTechnologies), and a mixture of any such media. A medium may or may notcontain blood serum. A medium may optionally contain one or more serumsubstitutes selected from among, for example, albumin, transferrin,Knockout Serum Replacement (KSR) (an FBS serum substitute used for EScell culture), N2 supplements (Invitrogen), B27 supplements(Invitrogen), fatty acid, insulin, collagen precursors, trace elements,2-mercaptoethanol, and 3′-thiol glycerol. In addition, a medium cancontain one or more substances selected from among, for example, lipids,amino acids, L-glutamine, Glutamax (Invitrogen), nonessential aminoacids, vitamins, growth factors, low-molecular-weight compounds,antibiotics, antioxidants, pyruvic acids, buffer agents, and inorganicsalts. A medium mixture of DMEM and Ham's F12 supplemented withGlutamax, B27, N2, 3′-thiol glycerol, and ascorbic acid is preferable.

In this step, the cells obtained in the previous step (i.e., the step ofculture in a medium containing a BMP inhibitor and a TGFβ inhibitor) arecultured in a medium prepared by supplementing the basal medium withBMP4, retinoic acid, and a GSK3β inhibitor.

The term “BMP4” used herein refers to a protein encoded by thepolynucleotide shown in the NCBI Accession Number NM_001202, NM_130850,or NM_130851, and it may be in an active form resulting from cleavage bya protease.

The BMP4 concentration in a medium is not particularly limited. Forexample, such concentration may be 10 ng/ml, 20 ng/ml, 30 ng/ml, 40ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml,900 ng/ml, or 1 μg/ml, although the concentration is not limitedthereto. The concentration is preferably 100 ng/ml.

While all-trans retinoic acid (ATRA) is exemplified as retinoic acid,artificially modified retinoic acid that retains functions of naturallyoccurring retinoic acid may be used. Examples thereof include4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]-benzoicacid (AM580) (Tamura, K. et al., Cell Differ. Dev., 32: 17-26, 1990),4-[(1E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]-benzoicacid (TTNPB) (Strickland, S., et al., Cancer Res., 43: 5268-5272, 1983),retinol palmitate, retinol, retinal, 3-dehydroretinoic acid,3-dehydroretinol, 3-dehydroretinal, and compounds described in Abe, E.,et al., Proc. Natl. Acad. Sci., (U.S.A.) 78: 4990-4994, 1981; Schwartz,E. L.et al., Proc. Am. Assoc. Cancer Res., 24: 18, 1983; Tanenaga, K. etal., Cancer Res., 40: 914-919, 1980.

The retinoic acid concentration in a medium is not particularly limited.For example, such concentration can be 1 nM, 5 nM, 10 nM, 15 nM, 20 nM,25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM,300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1 μM,although the concentration is not limited thereto. The concentration ispreferably 50 nM.

The GSK3β inhibitor as described above can be used in this step, and theGSK3β inhibitor is preferably CHIR99021. In this step, the CHIR99021concentration in a medium is, for example, 1 nM, 10 nM, 50 nM, 100 nM,500 nM, 750 nM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM,5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM,or 50 μM, although the concentration is not limited thereto. In thisstep, the concentration is preferably 2.5 μM.

In this step, culture may be conducted in a culture vessel treated witha coating agent. A coating agent may be a naturally occurring orartificially synthesized extracellular matrix. Examples thereof includeBD Matrigel, collagen, gelatin, laminin, heparan sulfate proteoglycan,entactin, and a combination of any thereof, with Matrigel beingpreferable.

This step may be implemented by exchanging the cell culture mediumobtained in the previous step with the medium described above.Alternatively, cells may be detached and reseeded in a culture vessel.When cells are to be detached, particular cells may be selected, and,for example, SOX2-, SOX17-, and/or FOXA2-positive cells may be selectedand used in this step. This method is preferably implemented by means ofmedia exchange.

When the step comprises a process of cell detachment, an ROCK inhibitormay be added to a medium, so as to inhibit pluripotent stem cell deathcaused by detachment.

Concerning culture conditions, culture is conducted at about 30□C to40□C, and preferably at about 37□C, although the temperature is notlimited thereto. Culture is conducted under an atmosphere of aircontaining CO₂, and the CO₂ concentration is preferably about 2% to 5%.

The culture period is not particularly limited because long-term culturewould not cause any problems. For example, the culture period may be atleast 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.The culture period is preferably at least 4 days, and more preferably 4days.

-   [Step of Culture in a Medium Containing FGF10]

A medium used in this step can be prepared from a medium used for animalcell culture as a basal medium. Examples of basal media include IMDMmedium, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEMmedium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium,RPMI 1640 medium, Fischer's medium, Neurobasal Medium (LifeTechnologies), and a mixture of any such media. A medium may or may notcontain blood serum. A medium may optionally contain one or more serumsubstitutes selected from among, for example, albumin, transferrin,Knockout Serum Replacement (KSR) (an FBS serum substitute used for EScell culture), N2 supplements (Invitrogen), B27 supplements(Invitrogen), fatty acid, insulin, collagen precursors, trace elements,2-mercaptoethanol, and 3′-thiol glycerol. In addition, a medium cancontain one or more substances selected from among, for example, lipids,amino acids, L-glutamine, Glutamax (Invitrogen), nonessential aminoacids, vitamins, growth factors, low-molecular-weight compounds,antibiotics, antioxidants, pyruvic acids, buffer agents, and inorganicsalts. A medium mixture of DMEM and Ham's F12 supplemented withGlutamax, B27, N2, 3′-thiol glycerol, and ascorbic acid is preferable.

In this step, the cells obtained in the previous step (i.e., the step ofculture in a medium containing BMP4, retinoic acid, and a GSK3βinhibitor) are cultured in a medium prepared by supplementing the basalmedium with FGF10.

The term “FGF10 ” used herein refers to a protein encoded by thepolynucleotide shown in the NCBI Accession Number NM_004465, and it maybe in an active form resulting from cleavage by a protease. Such FGF10is commercially available from, for example, Life Technologies or Wako.

The FGF10 concentration in a medium is not particularly limited. Forexample, such concentration may be 10 ng/ml, 20 ng/ml, 30 ng/ml, 40ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml,900 ng/ml, or 1 μg/ml, although the concentration is not limitedthereto. The concentration is preferably 100 ng/ml.

In this step, culture may be conducted in a culture vessel treated witha coating agent. A coating agent may be a naturally occurring orartificially synthesized extracellular matrix. Examples thereof includeBD Matrigel, collagen, gelatin, laminin, heparan sulfate proteoglycan,entactin, and a combination of any thereof, with Matrigel beingpreferable.

This step may be implemented by exchanging the cell culture mediumobtained in the previous step with the medium described above.Alternatively, cells may be detached and reseeded in a culture vessel.When cells are to be detached, particular cells may be selected, and,for example, NKX2-1- and/or FOXA2-positive cells may be selected andused in this step. This method is preferably implemented by means ofmedia exchange.

When the step comprises a process of cell detachment, an ROCK inhibitormay be added to a medium, so as to inhibit pluripotent stem cell deathcaused by detachment.

Concerning culture conditions, culture is conducted at about 30□C to40□C, and preferably at about 37□C, although the temperature is notlimited thereto. Culture is conducted under an atmosphere of aircontaining CO₂, and the CO₂ concentration is preferably about 2% to 5%.

The culture period is not particularly limited because long-term culturewould not cause any problems. For example, the culture period may be atleast 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, or 14 days. The culture period is preferablyat least 7 days, and more preferably 7 days.

-   [Step of Culture in a Medium Containing a Steroid Drug, a cAMP    Derivative, a Phosphodiesterase Inhibitor, and KGF]

A medium used in this step can be prepared from a medium used for animalcell culture as a basal medium. Examples of basal media include IMDMmedium, Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEMmedium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium,RPMI 1640 medium, Fischer's medium, Neurobasal Medium (LifeTechnologies), and a mixture of any such media. A medium may or may notcontain blood serum. A medium may optionally contain one or more serumsubstitutes selected from among, for example, albumin, transferrin,Knockout Serum Replacement (KSR) (an FBS serum substitute used for EScell culture), N2 supplements (Invitrogen), B27 supplements(Invitrogen), fatty acid, insulin, ITS Premix, collagen precursors,trace elements, 2-mercaptoethanol, and 3′-thiol glycerol. In addition, amedium can contain one or more substances selected from among, forexample, lipids, amino acids, L-glutamine, Glutamax (Invitrogen),nonessential amino acids, vitamins, growth factors, low-molecular-weightcompounds, antibiotics, antioxidants, pyruvic acids, buffer agents, andinorganic salts. Ham's F12 medium containing albumin, buffer agents(e.g., HEPES), calcium chloride, ITS Premix, and antibiotics ispreferable.

In this step, the cells obtained in the previous step (i.e., the step ofculture in a medium containing FGF10) are cultured in a medium preparedby supplementing the basal medium with a steroid drug, a cAMPderivative, a phosphodiesterase inhibitor, and KGF.

The term “steroid drug” used herein refers to a steroidalanti-inflammatory drug, such as glucocorticoid or a synthetic derivativethereof. Specific examples thereof include hydrocortisone,hydrocortisone succinate, prednisolone, methylprednisolone,methylprednisolone succinate, triamcinolone, triamcinolone acetonide,dexamethasone, and betamethasone.

A steroid drug that can be preferably used in the present invention isdexamethasone. The dexamethasone concentration in a medium is notparticularly limited. For example, such concentration may be 1 nM, 5 nM,10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM,200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1 μM,although the concentration is not limited thereto. The concentration ispreferably 50 nM.

The term “cAMP derivative” used herein refers to a compound with amodified cyclic AMP substituent. Examples thereof include cyclicadenosine monophosphate (cAMP), 8-bromo cyclic adenosine monophosphate(8-Br-cAMP or 8Br-cAMP), 8-chloro-cyclic adenosine monophosphate(8-Cl-cAMP), 8-(4-chlorophenylthio)cyclic adenosine monophosphate(8-CPT-cAMP), and dibutyryl cyclic adenosine monophosphate (DB-cAMP).

A cAMP derivative that can be preferably used in the present inventionis 8-Br-cAMP. The concentration of 8-Br-cAMP in a medium is notparticularly limited. For example, such concentration can be 1 μM, 5 μM,10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM,200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, or 1 mM,although the concentration is not limited thereto. The concentration ispreferably 100 μM.

The term “phosphodiesterase inhibitor” used herein refers to a compoundthat inhibits phosphodiesterase (PDE), so as to increase theconcentration of cAMP or cGMP in the cells. Examples thereof include1,3-dimethylxanthine, 6,7-dimethoxy-1-(3,4-dimethoxybenzyl)isoquinoline,4-{[3′,4′-(methylenedioxy)benzyl]amino}-6-methoxyquinazoline,8-methoxymethyl-3-isobutyl-1-methylxanthine, and3-isobutyl-1-methylxanthine (IBMX).

A phosphodiesterase inhibitor that can be preferably used in the presentinvention is IBMX. The IBMX concentration in a medium is notparticularly limited. For example, such concentration can be 1 μM, 5 μM,10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM,200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, or 1 mM,although the concentration is not limited thereto. The concentration ispreferably 100 μM.

The term “KGF” used herein refers to a protein encoded by thepolynucleotide shown in the NCBI Accession Number NM_002009, and it maybe in an active form resulting from cleavage by a protease. Such KGF iscommercially available from, for example, Wako.

The concentration of KGF in a medium is not particularly limited. Forexample, such concentration can be 10 ng/ml, 20 ng/ml, 30 ng/ml, 40ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 700 ng/ml, 800 ng/ml,900 ng/ml, or 1 μg/ml, although the concentration is not limitedthereto. The concentration is preferably 100 ng/ml.

In this step, culture may be conducted in a culture vessel treated witha coating agent. A coating agent may be a naturally occurring orartificially synthesized extracellular matrix. Examples thereof includeBD Matrigel, collagen, gelatin, laminin, heparan sulfate proteoglycan,entactin, and a combination of any thereof, with Matrigel beingpreferable.

This step may be implemented by exchanging the cell culture mediumobtained in the previous step with the medium described above.Alternatively, cells may be detached and reseeded in a culture vessel.When cells are to be detached, particular cells may be selected, and,for example, NKX2-1-positive cells may be selected and used in thisstep. This method is preferably implemented by means of media exchange.

When the step comprises a process of cell detachment, an ROCK inhibitormay be added to a medium, so as to inhibit pluripotent stem cell deathcaused by detachment.

Concerning culture conditions, culture is conducted at about 30□C to40□C, and preferably at about 37□C, although the temperature is notlimited thereto. Culture is conducted under an atmosphere of aircontaining CO₂, and the CO₂ concentration is preferably about 2% to 5%.

The culture period is not particularly limited because long-term culturewould not cause any problems. For example, the culture period may be atleast 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.The culture period is preferably at least 4 days, and it is morepreferably 4 days.

[Three-Dimensional Culture]

The present invention provides a method for three-dimensional culture ofalveolar epithelial progenitor cells for further maturation of alveolarepithelial progenitor cells. According to the present invention,three-dimensional culture is carried out by subjecting cells to floatculture in the form of cell masses (i.e., spheroids). According to thepresent invention, three-dimensional culture can be carried out with theuse of, for example, Cell Culture Inserts provided by BD.

According to the present invention, three-dimensional culture may beconducted in the presence of other cell species. Examples of other cellspecies that may be used include human pulmonary fibroblasts and humanfetal pulmonary fibroblasts. Such cells are commercially available from,for example, American Type Culture Collection (ATCC) and DV Biologics.

The medium used for three-dimensional culture according to the presentinvention may be a medium that is used in the step of culture conductedin a medium containing a steroid drug, a cAMP derivative, aphosphodiesterase inhibitor, and KGF. Use of a medium supplemented withan extracellular matrix may be preferable. The ratio of the volume ofthe extracellular matrix to the volume of the medium is not particularlylimited. For example, these substances can be mixed at a ratio of 5:1,4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5. In the present invention, anextracellular matrix is a supramolecular structure that exists outsidethe cell, and it may be a naturally occurring or artificial(recombinant) structure. Examples thereof include substances such ascollagen, proteoglycan, fibronectin, hyaluronic acid, tenascin,entactin, elastin, fibrillin, and laminin, and fragments thereof. Theseextracellular matrices may be used in combination. For example,extracellular matrices may be prepared from cells such as BD Matrigel™.An example of an artificial structure is a laminin fragment.

The period for three-dimensional culture is not particularly limitedbecause long-term culture would not cause any problems. For example, theculture period may be at least 5 days, 6 days, 7 days, 8 days, 9 days,10 days, 11 days, or 12 days. The culture period is preferably at least10 days, and particularly preferably 10 days, 11 days, or 12 days.

-   [Pluripotent Stem Cells]

Pluripotent stem cells that can be used in the present invention arestem cells that have the potential to differentiate into any types ofcells existing in organisms (i.e., pluripotency) and have the potentialto grow. Examples thereof include embryonic stem cells (ES cells),nuclear transfer-derived embryonic stem cells from cloned embryos (ntEScells), germline stem cells (GS cells), embryonic germ cells (EG cells),induced pluripotent stem cells (iPS cells), and pluripotent cellsderived from cultured fibroblasts and myeloid stem cells (Muse cells).In the present invention, the use of iPS cells or Muse cells ispreferable because cells of interest can be obtained without destroyingembryos.

(A) Embryonic Stem Cells

ES cells are pluripotent stem cells having the potential to grow throughautoreproduction, and they are established from embryoblasts of earlyembryos (e.g., blastocysts) of mammalians such as humans or mice.

ES cells are embryo-derived stem cells originating from embryoblasts ofblastocysts, which are embryos after the 8-cell stage and the morulastage of fertilized eggs. Such ES cells have the potential todifferentiate into any types of cells constituting an adult; that is,so-called pluripotency and potential to grow through autoreproduction.ES cells were discovered in mice in 1981 (M. J. Evans and M. H. Kaufman,1981, Nature 292: 154-156). Thereafter, ES cells of primates, such ashumans and monkeys, were also established (J. A. Thomson, et al., 1998,Science 282: 1145-1147; J. A. Thomson, et al., 1995, Proc. Natl. Acad.Sci., U.S.A., 92: 7844-7848; J. A. Thomson, et al., 1996, Biol. Reprod.,55: 254-259; J. A. Thomson and V. S. Marshall, 1998, Curr. Top. Dev.Biol., 38: 133-165).

ES cells can be established by extracting embryoblasts from blastocystsof fertilized eggs of target animals and culturing the embryoblasts infibroblast feeders. Cells can be maintained via subculture with the useof a medium supplemented with substances such as leukemia inhibitoryfactors (LIF) and basic fibroblast growth factors (bFGF). Human andmonkey ES cells can be established and maintained by the methodsdescribed in, for example, U.S. Pat. No. 5,843,780; Thomson J. A., etal., 1995, Proc. Natl. Acad. Sci., U.S.A., 92: 7844-7848; Thomson, J.A., et al., 1998, Science 282: 1145-1147; H. Suemori et al., 2006,Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al., 2006,Proc. Natl. Acad. Sci. U.S.A., 103:9554-9559; H. Suemori et al., 2001,Dev. Dyn., 222:273-279; H. Kawasaki et al., 2002, Proc. Natl. Acad. Sci.U.S.A., 99:1580-1585; and Klimanskaya I, et al., 2006, Nature 444:481-485.

Human ES cells can be maintained with the use of a medium for theproduction of ES cells, such as a DMEM/F-12 medium supplemented with 0.1mM 2-mercaptoethanol, 0.1 mM nonessential amino acids, 2 mM L-glutamicacid, 20% KSR, and 4 ng/ml bFGF, at 37□C in the presence of 5% CO₂ in amoist atmosphere (H. Suemori, et al., 2006, Biochem. Biophys. Res.Commun., 345: 926-932). It is necessary that ES cells be subjected tosubculture every 3 or 4 days. Subculture can be carried out with the useof 0.25% trypsin and 0.1 mg/ml collagenase IV in PBS containing 1 mMCaCl₂ and 20% KSR.

In general, ES cells can be selected via real-time PCR using theexpression of a gene marker such as alkaline phosphatase, Oct-3/4, orNanog as an indicator. When human ES cells are to be selected, inparticular, the expression of a gene marker such as OCT-3/4, NANOG, orECAD can be employed as an indicator (E. Kroon et al., 2008, Nat.Biotechnol., 26: 443-452).

Human ES cells (e.g., WA01 (H1) and WA09 (H9)) are available from theWiCell Research Institute, and KhES-1, KhES-2, and KhES-3 are availablefrom the Institute for Frontier Medical Sciences, Kyoto University(Kyoto, Japan).

(B) Germline Stem Cells

Germline stem cells are spermary-derived pluripotent stem cells thatserve as sources for spermatogenesis. As with the case of ES cells,germline stem cells can be differentiated into various types of cells.For example, germline stem cells may be implanted into mouseblastocysts, so that chimeric mice may be produced (M. Kanatsu-Shinoharaet al., 2003, Biol. Reprod., 69: 12-616; K. Shinohara et al., 2004,Cell, 119: 1001-1012). Germline stem cells are capable ofautoreproduction in a medium containing glial cell line-derivedneurotrophic factors (GDNF). In addition, germline stem cells can beobtained by repeating subculture under the same culture conditions aswith those used for ES cells (Masanori Takebayashi et al., 2008,Experimental Medicine, Vol. 26, No. 5 (extra edition), pp. 41-46,Yodosha, Tokyo, Japan).

(C) Embryonic Germ Cells

As with ES cells, embryonic germ cells are pluripotent cells that areestablished from primordial germ cells during the prenatal period.Embryonic germ cells can be established by culturing primordial germcells in the presence of substances such as LIF, bFGF, or stem cellfactors (Y. Matsui et al., 1992, Cell, 70: 841-847; J. L. Resnicket al.,1992, Nature, 359: 550-551).

(D) Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells can be prepared by introducingparticular reprogramming factors into somatic cells in the form of DNAor proteins. iPS cells are artificial stem cells derived from somaticcells that have substantially the same properties as ES cells, such aspluripotency and potential to grow through autoreproduction (K.Takahashi and S. Yamanaka, 2006, Cell, 126: 663-676; K. Takahashi etal., 2007, Cell, 131: 861-872; J. Yu et al., 2007, Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol., 26: 101-106, 2008; WO2007/069666). Reprogramming factors may be 4composed of genes that areexpressed specifically in ES cells, gene products or non-cording RNAthereof, or genes that play key roles in maintenance of theundifferentiated state of ES cells, gene products or non-coding RNAthereof, or low-molecular-weight compounds. Examples of genes includedin reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17,K1f4, K1f2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2,Tcll, beta-catenin, Lin28b, Sa111, Sa114, Esrrb, Nr5a2, Tbx3, and Glis1.Such reprogramming factors may be used alone or in combination. Examplesof combinations of reprogramming factors are described in WO2007/069666, WO 2008/118820, WO 2009/007852, WO 2009/032194, WO2009/058413, WO 2009/057831, WO 2009/075119, WO 2009/079007, WO2009/091659, WO 2009/101084, WO 2009/101407, WO 2009/102983, WO2009/114949, WO 2009/117439, WO 2009/126250, WO 2009/126251, WO2009/126655, WO 2009/157593, WO 2010/009015, WO 2010/033906, WO2010/033920, WO 2010/042800, WO 2010/050626, WO 2010/056831, WO2010/068955, WO 2010/098419, WO 2010/102267, WO 2010/111409, WO2010/111422, WO 2010/115050, WO 2010/124290, WO 2010/147395, WO2010/147612, Huangfu D, et al., 2008, Nat. Biotechnol., 26: 795-797, ShiY, et al., 2008, Cell Stem Cell, 2: 525-528, Eminli S, et al., 2008,Stem Cells, 26: 2467-2474, Huangfu D, et al., 2008, Nat. Biotechnol.,26: 1269-1275, Shi Y, et al., 2008, Cell Stem Cell, 3, 568-574, Zhao Y,et al., 2008, Cell Stem Cell, 3: 475-479, Marson A, 2008, Cell StemCell, 3, 132-135, Feng B., et al., 2009, Nat Cell Biol., 11: 197-203, R.L. Judson et al., 2009, Nat. Biotech., 27: 459-461, Lyssiotis C A, etal., 2009, Proc. Natl. Acad. Sci., U.S.A. 106: 8912-8917, Kim J B, etal., 2009, Nature, 461: 649-643, Ichida, J. K., et al., 2009, Cell StemCell, 5: 491-503, Heng J. C. et al., 2010, Cell Stem Cell, 6: 167-74,Han J, et al., 2010, Nature, 463: 1096-100, Mali P, et al., 2010, andStem Cells, 28: 713-720, Maekawa M, et al., 2011, Nature, 474: 225-9.

Factors that are used to enhance cell establishment efficiency arewithin the scope of the reprogramming factors described above. Examplesthereof include: histone deacetylase (HDAC) inhibitors, such aslow-molecular-weight inhibitors, including valproic acid (VPA),trichostatin A, sodium butyrate, MC 1293, and M344, and nucleicacid-based expression inhibitors, including siRNAs and shRNAs againstHDAC (e.g., HDAC1 siRNA Smartpoor (Millipore) and HuSH 29mer shRNAconstructs against HDAC1 (OriGene)); MEK inhibitors (e.g., PD184352,PD98059, U0126, SL327, and PD0325901); glycogen synthase kinase-3inhibitors (e.g., Bio and CHIR99021); DNA methyltransferase inhibitors(e.g., 5-azacytidine); histone methyltransferase inhibitors (e.g.,low-molecular-weight inhibitors, such as BIX-01294, and nucleicacid-based expression inhibitors against Suv39h1, Suv39h2, SetDB1 andG9a, such as siRNAs and shRNAs); an L-channel calcium agonist (e.g.,Bayk8644); butyric acid, TGFβ, and ALKS inhibitors (e.g., LY364947,SB431542, 616453, and A-83-01); p53 inhibitors (e.g., siRNA and shRNAagainst p53); ARID3A inhibitors (e.g., siRNA and shRNA against ARID3A),miRNA, such as miR-291-3p, miR-294, miR-295, and mir-302, Wnt signaling(e.g., soluble Wnt3a), neuro-peptide Y, prostaglandins (e.g.,prostaglandin E2 and prostaglandinJ2), hTERT, SV40LT, UTF1, IRX6, GLIS1,PITX2, and DMRTB1. Factors that are used for the improvement ofestablished efficiency are not particularly distinguished fromreprogramming factors.

When reprogramming factors are in the form of proteins, for example,they may be introduced into somatic cells by a technique such aslipofection, fusion with cell-permeable peptides (e.g., HIV-derived TATand polyarginine), or microinjection.

In contrast, reprogramming factors in the form of DNA can be introducedinto somatic cells by a technique involving the use of a vector such asa virus, plasmid, or artificial chromosome vector, lipofection, atechnique involving the use of a liposome, or microinjection, forexample. Examples of virus vectors include retrovirus vectors,lentivirus vectors (Cell, 126, pp. 663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp. 1917-1920, 2007), adenovirus vectors(Science, 322, 945-949, 2008), adeno-associated virus vectors, andSendai virus vectors (WO 2010/008054). Examples of artificial chromosomevectors include human artificial chromosome (HAC) vectors, yeastartificial chromosome (YAC) vectors, and bacterial artificial chromosome(BAC, PAC) vectors. Plasmids for mammalian animal cells can be used(Science, 322: 949-953, 2008). Vectors can comprise regulatorysequences, such as promoters, enhancers, ribosome-binding sequences,terminators, or polyadenylation sites, so that nuclear reprogrammingsubstances can express. In addition, vectors can comprise selectionmarker sequences, such as drug tolerance genes (e.g., kanamycintolerance genes, ampicillin tolerance genes, and puromycin tolerancegenes), thymidine kinase genes, or diphtheria toxin genes, and reportergene sequences, such as green fluorescent proteins (GFP),P-glucuronidase (GUS), or FLAG, according to need. The vector maycomprise LoxP sequences in positions downstream and upstream of a geneencoding an reprogramming factor or a gene encoding a promoter and anreprogramming factor binding thereto, so as to eliminate such gene afterthe vector is introduced into somatic cells.

When reprogramming factors are in the form of RNA, for example, they maybe introduced into somatic cells by a technique such as lipofection ormicroinjection. Alternatively, RNA comprising 5-methylcytidine andpseudouridine (TriLink Biotechnologies) incorporated therein may beused, so as to suppress degradation (Warren L, 2010, Cell Stem Cell 7:618-630).

Examples of culture media used for iPS cell induction include DMEMcontaining 10% to 15% FBS, a DMEM/F12 or DME medium (such medium mayadequately contain, for example, LIF, penicillin/streptomycin,puromycin, L-glutamine, nonessential amino acids, and(3-mercaptoethanol), commercially available culture media (e.g., amedium for mouse ES cell culture; TX-WES medium, Thrombo X), a mediumfor primate ES cell culture (a medium for primate ES/iPS cell culture,ReproCELL Incorporated), and a serum-free medium (mTeSR, StemcellTechnology).

For example, somatic cells are brought into contact with reprogrammingfactors in a 10% FBS-containing DMEM or DMEM/F12 medium, culture isconducted at 37□C in the presence of 5% CO₂ for about 4 to 7 days, andthe cells are reseeded in feeder cells (e.g., mitomycin C-treated STOcells or SNL cells). Culture is reinitiated in a medium forbFGF-containing primate ES cell culture about 10 days after the somaticcells are first brought into contact with the reprogramming factors, andiPS-like colonies can then be formed at least about 30 to 45 days aftersuch contact.

Alternatively, culture may be conducted in a 10% FBS-containing DMEMmedium (this medium can further contain LIF, penicillin/streptomycin,puromycin, L-glutamine, nonessential amino acids, (3-mercaptoethanol, orthe like, according to need) in feeder cells (e.g., mitomycin C-treatedSTO cells or SNL cells) at 37□C in the presence of 5% CO₂, and ES-likecolonies can then be formed at least about 25 to 30 days later.Alternatively, use of the somatic cells to be reprogrammed instead offeeder cells is preferable (Takahashi K, et al., 2009, PLoS One, 4:e8067 or WO 2010/137746), or use of an extracellular matrix (e.g.,laminin-5 (WO 2009/123349) and Matrigel (BD)) is preferable.

In addition, culture may be conducted with the use of a serum-freemedium (Sun N, et al., 2009, Proc. Natl. Acad. Sci., U.S.A. 106:15720-15725). In order to enhance cell establishment efficiency, iPScells may be established under low-oxygen conditions (oxygenconcentration of 0.1 % to 15%) (Yoshida Y, et al., 2009, Cell Stem Cell,5: 237-241 or WO 2010/013845).

During the culture, medium exchange is initiated 2 days after theinitiation of culture, and the medium is exchanged with fresh mediumonce a day. The number of somatic cells used for nuclear reprogrammingis not limited, and it is about 5×10³ to about 5×10⁶ cells per 100 cm²of a culture dish.

iPS cells can be selected in accordance with the configuration of theformed colonies. When drug tolerance genes that express in associationwith genes that express upon reprogramming of somatic cells (e.g.,Oct3/4 and Nanog) are introduced as marker genes, in contrast, culturecan be conducted in a medium containing corresponding drugs (i.e., aselection medium). Thus, established iPS cells can be selected. Whenmarker genes are fluorescent protein genes, fluorescent microscopicobservation may be carried out. When marker genes are luminescent enzymegenes, luminescent substrates may be added. When marker genes arechromogenic enzyme genes, chromogenic substrates may be added. Thus, iPScells can be selected.

The term “somatic cells” used herein refers to any animal cells exceptfor germline cells or pluripotent cells such as egg cells, oocytes, andES cells (preferably mammalian animal cells, including those of humans).Examples of somatic cells include, but are not limited to, embryonic(fetal) somatic cells, neonatal (fetal) somatic cells, and maturehealthy or affected somatic cells. Somatic cells may be primary-culturedcells, subcultured cells, or established cells. Specific examples ofsomatic cells include: (1) tissue stem cells, such as neural stem cells,hematopoietic stem cells, mesenchymal stem cells, and dental pulp stemcells (i.e., somatic stem cells); (2) tissue progenitor cells; (3)differentiated cells, such as lymphocytes, epidermic cells, endothelialcells, muscle cells, fibroblasts (e.g., skin cells), hair cells, hepaticcells, gastric mucosal cells, intestinal cells, splenic cells,pancreatic cells (e.g., pancreatic exocrine cells), brain cells,pneumocytes, nephrocytes, and adipocytes.

When iPS cells are used as materials for transplantation, use of somaticcells having the same or substantially the same HLA genotype as that ofa recipient is preferable, so that rejection would not occur. When HLAgenotypes are “substantially the same,” such HLA genotypes areconcordant with each other to the extent that an immunosuppressive agentis able to suppress immune responses to the transplanted cells. Forexample, such somatic cells have HLA genotypes exhibiting concordance in3 loci; i.e., HLA-A, HLA-B, and HLA-DR, or in 4 loci; i.e., HLA-A,HLA-B, HLA-DR, and HLA-C.

(E) Nuclear Transfer-Derived ES Cells from Cloned Embryos

“nt ES cells” are nuclear transfer-derived ES cells produced from clonedembryos, and such ES cells have substantially the same properties asfertilized egg-derived ES cells (T. Wakayama et al., 2001, Science, 292:740-743; S. Wakayama et al., 2005, Biol. Reprod., 72: 932-936; J. Byrneet al., 2007, Nature, 450: 497-502). Specifically, nuclear transfer EScells (i.e., nt ES cells) are ES cells that are established fromembryoblasts of blastocysts derived from cloned embryos resulting fromsubstitution of an unfertilized egg nucleus with a somatic cell nucleus.nt ES cells are produced by the technique of nuclear transfer (J. B.Cibelli et al., 1998, Nature Biotechnol., 16: 642-646) in combinationwith the technique of ES cell production (Kiyoka Wakayama et al., 2008,Experimental Medicine, Vol. 25, No. 5 (extra edition), pp. 47-52). Inthe case of nuclear transfer, somatic cell nuclei are injected intoenucleated unfertilized eggs of mammalian animals, and culture isconducted for several hours. Thus, such cells can be reprogrammed.

(F) Multilineage-Differentiating Stress Enduring Cells (Muse Cells)

Muse cells are pluripotent stem cells produced by the method describedin WO 2011/007900. More specifically, Muse cells are pluripotent cellsthat are obtained by treating fibroblasts or myeloid interstitial cellswith tryp sin for a long period of time (preferably for 8 hours or 16hours) and conducting float culture. Such cells are positive for SSEA-3and CD105.

[Kit for Inducing Pluripotent Stem Cells to Differentiate into AlveolarEpithelial Progenitor Cells]

The present invention provides a kit used for inducing pluripotent stemcells to differentiate into alveolar epithelial progenitor cells orproducing alveolar epithelial progenitor cells from pluripotent stemcells. The kit may comprise growth factors, compounds, a medium, a celldetachment solution, and an agent for coating the culture vessel usedfor the induction of differentiation. The kit may further comprisedocuments and/or instructions describing the procedure for the inductionof differentiation.

[Method for Selecting Alveolar Epithelial Progenitor Cells]

In the present invention, alveolar epithelial progenitor cells mayconstitute a population of cells including alveolar epithelialprogenitor cells. In the present invention, a population of cellsincluding alveolar epithelial progenitor cells preferably includes 50%,60%, 70%, 80%, or 90% or more alveolar epithelial progenitor cells.

Accordingly, the present invention provides a method for extractingalveolar epithelial progenitor cells. The cells to be extracted may bealveolar epithelial progenitor cells obtained by the method describedabove or cells obtained in the process for producing the same, whichtakes place after the completion of Step (3) of culture in a mediumcontaining BMP4, retinoic acid, and a GSK3β inhibitor or Step (4) ofculture in a medium containing FGF10. Alveolar epithelial progenitorcells can be extracted with the use of a reagent having CPM-specificaffinity. While CPM had been known as a marker of adult type I alveolarepithelial cells, it was not previously known that CPM was expressed inprogenitor cells during development. That is, use of CPM as a surfacemarker in the same manner as with NKX2-1 as a marker of alveolarepithelial progenitor cells has been found for the first time throughthe present invention.

Examples of reagents having specific affinity that can be used in thepresent invention include antibodies, aptamers, peptides, and compoundsthat specifically recognize the substances of interest, with antibodiesor fragments thereof being preferable.

In the present invention, antibodies may be polyclonal or monoclonalantibodies. Such antibodies can be prepared in accordance withtechniques well known in the art (Current protocols in MolecularBiology, Ausubel et al. (editors), 1987, John Wiley and Sons(publisher), Section 11.12-11.13). When the antibodies of the presentinvention are polyclonal antibodies, specifically, proteins encoded byCPM expressed in E. coli or mammalian cells and purified, oligopeptideshaving partial amino acid sequences, or glycolipids may be purified inaccordance with conventional techniques, nonhuman animals such asrabbits may be immunized therewith, and antibodies of interest can beobtained from sera of the immunized animals in accordance with aconventional technique. In the case of monoclonal antibodies, incontrast, antibodies of interest can be obtained from hybridoma cellsprepared via fusion of spleen cells obtained from immunized nonhumananimals to myeloma cells (Current protocols in Molecular Biology,Ausubel et al. (editors), 1987, John Wiley and Sons (publisher), Section11.4-11.11). Examples of antibody fragments include a part of anantibody (e.g., an Fab fragment) and a synthetic antibody fragment(e.g., a single-stranded Fv fragment, ScFv). Antibody fragments, such asFab and F(ab)2 fragments, can be prepared by a genetic engineeringtechnique well known in the art. For example, antibodies reacting withCPM can be obtained from Leica Microsystems.

In order to recognize or separate cells that express CPM, reagentshaving relevant affinity may be bound or conjugated to substances thatenable detection, such as a fluorescent label, a radioactive label, achemoluminescent label, an enzyme, biotin, or streptoavidin, orsubstances that enable isolation and extraction, such as Protein A,Protein G, beads, or magnetic beads.

Alternatively, reagents having relevant affinity may be indirectlylabeled. Labeling may be carried out in accordance with varioustechniques known in the art. For example, pre-labeled antibodies(secondary antibodies) that specifically bind to the antibodiesdescribed above may be used.

Alveolar epithelial progenitor cells can be extracted by, for example, amethod comprising conjugating particles to a reagent having relevantaffinity in order to precipitate the cells, a method involving the useof magnetic beads to select the cells with the aid of magnetism (e.g.,MACS), a method involving the use of a cell sorter with the aid of afluorescent label, or a method involving the use of a support upon whichantibodies or the like are immobilized (e.g., a cell enrichment column).

[Agents for Treatment of Pulmonary Alveolar Diseases]

The alveolar epithelial progenitor cells obtained in the presentinvention can be administered to patients afflicted with diseases thatdestroy the pulmonary alveolus in the form of pharmaceuticalpreparations. The alveolar epithelial progenitor cells are prepared intothe form of a sheet, and the sheet may be applied to the alveolarepithelium of a patient. Alternatively, the alveolar epithelialprogenitor cells may be suspended in physiological saline or the like,and the suspension may then be directly implanted in the pulmonaryalveolus of the patient. Accordingly, the present invention provides anagent for treatment of pulmonary alveolar diseases comprising alveolarepithelial progenitor cells obtained from pluripotent stem cells in themanner described above.

In the present invention, the number of alveolar epithelial progenitorcells contained in the agent for treatment of pulmonary alveolardiseases is not particularly limited, provided that the transplantedgrafts are able to survive. The number of the cells may be adequatelyadjusted in accordance with lesion size or body size.

EXAMPLES

Hereafter, the present invention is described in greater detail withreference to the examples, although the technical scope of the presentinvention is not limited to these examples.

[iPS Cell Culture]

Human iPS cells (201B7) were provided by Professor Yamanaka at KyotoUniversity and cultured in accordance with a conventional technique(Takahashi K, et al. Cell, 131: 861-872, 2007). In accordance with themethod described in Mae S., et al, Nat. Commun., 4: 1367, 2013,according to a gene knock-in technique, SFTPC-reporter 201B7 wasproduced by introducing an EGFP sequence into a site downstream of theSFTPC initiation codon of the human iPS cells (201B7).

-   [Induction of Alveolar Epithelial Progenitor Cells]

FIG. 1 shows a scheme for producing alveolar epithelial progenitor cellsfrom pluripotent stem cells such as iPS cells.

The alveolar epithelial progenitor cells were induced by detaching humaniPS cells with the use of Accutase, seeding the cells in a 24-well platecoated with Matrigel at 2.0×10⁵ cells/well or in a 6-well plate coatedwith Matrigel at 9.6×10⁵ cells/well, and conducting culture under theconditions described below (FIGS. 1A and 1B).

(Step 1)

The seeded cells (Day 0) were cultured in basal medium 1 (RPMI1640(Nacalai Tesque) containing 2% B27 (Life Technologies) and a 0.5%penicillin/streptomycin stock solution (Life Technologies)) supplementedwith 100 ng/ml Activin A (R&D Systems), 1 μM CHIR99021 and 10 μMY-27632. On the following day (Day 1), the medium was exchanged withbasal medium 1 containing 100 ng/ml Activin A, 1 μM CHIR99021, and 0.25mM NaB, the medium was exchanged with another medium under the sameconditions on the following day (Day 2) and 3 days later (Day 4), andculture was conducted for 5 days.

Alternatively, the seeded cells (Day 0) were cultured in basal medium 1supplemented with 100 ng/ml Activin A, 1 μM CHIR99021, and 10 μMY-27632. On the following day (Day 1), the medium was exchanged withbasal medium 1 containing 100 ng/ml Activin A, 1 μM CHIR99021, 10 μMY-27632, and 0.125 mM or 0.25 mM NaB, the medium was exchanged withbasal medium 1 containing 100 ng/ml Activin A, 1 μM CHIR99021, and 0.125mM or 0.25 mM NaB on the following day (Day 2), and the medium wasexchanged with another medium under the same conditions 3 days later(Day 4).

(Step 2)

The cells obtained in Step 1 (Day 6) were cultured in basal medium 2(DMEM/F12 medium (Life Technologies) containing 1% Glutamax supplement(Life Technologies), 2% B27 supplement, 1% N2 supplement (LifeTechnologies), 0.8% StemSure™ 50 mmol/l monothioglycerol solution(Wako), 50 μL-ascorbic acid (Sigma Aldrich), and 0.5%penicillin/streptomycin stock solution) supplemented with 200 ng/ml or100 ng/ml hNoggin (R&D Systems) and 10 μM SB-431542 for 4 days. In thiscase, the medium was exchanged with another medium under the sameconditions every other day.

(Step 3)

The cells obtained in Step 2 (Day 10) were cultured in basal medium 2containing 100 ng/ml hBMP4 (HumanZyme, Inc.), 0.05 μM all-trans retinoicacid (ATRA), and 2.5 μM CHIR99021 for 4 days. In this case, the mediumwas exchanged with another medium under the same conditions every otherday.

(Step 4)

The cells obtained in Step 3 (Day 14) were cultured in basal medium 2containing 100 ng/ml FGF10 (Wako) for 7 days. In this case, the mediumwas exchanged with another medium under the same conditions every otherday.

(Step 5)

After the media were exchanged, the cells obtained in Step 4 (Day 21)were cultured in basal medium 3 (Ham's F12 media (Wako) containing 3.33%BSA Fraction V Solution (7.5%) (Life Technologies), 15 mM HEPES (SigmaAldrich), 0.8 mM CaCl2 (Nacalai Tesque), 1% ITS Premix (BD), and 0.5%penicillin/streptomycin stock solution) containing 50 nM dexamethasone(Sigma Aldrich), 0.1 mM 8-Br-cAMP (Biolog Life Science Institute), 0.1mM 3-isobutyl-1-methylxanthine (IBMX) (Wako), and 100 ng/ml or 50 ng/mlKGF (Wako). Thereafter, the medium was exchanged with another mediumunder the same conditions every other day. The alveolar epithelialprogenitor cells obtained were analyzed 4 days later (Day 25).

-   [Cell Analysis]

(1) After the Completion of Step 3

After the completion of Step 3 (Day 14), the cells were subjected toimmunostaining so as to inspect CPM and NKX2-1 expression. As a result,cells positive for both such markers were identified (FIGS. 2A and 2B).In addition, CPM-positive cells were separated from the cells obtainedon Day 14 using MACS (Miltenyi Biotec) (FIGS. 3A and 3B), and theobtained cells were allowed to adhere to glass slides via cytospinning,followed by immunostaining. As a result, many thereof were found to alsobe positive for NKX2-1 (FIGS. 4A and 4B). The cells were analyzed usinga flow cytometer. As a result, 92% of the CPM-positive cells obtained byMACS were found to be NKX2-1-positive (FIGS. 5A and 5B). Also,CPM-positive cells identified via MACS were subjected to quantitativeRT-PCR, so as to assay CPM and NKX2-1 mRNA levels. As a result, sortingof CPM-positive cells was found to result in a remarkable increase inCPM and NKX2-1 mRNA levels (FIG. 6).

(2) After the Completion of Step 4

After the completion of Step 4 (Day 21), CPM-positive cells wereseparated from cells using MACS, and the CPM and NKX2-1 mRNA levels wereassayed via quantitative RT-PCR. As a result, sorting of CPM-positivecells was found to result in a remarkable increase in CPM and NKX2-1mRNA levels (FIG. 7).

(3) After the Completion of Step 5

After the completion of Step 5 (Day 25), CPM-positive cells wereseparated from cells using MACS, and the obtained cells were allowed toadhere to glass slides via cytospinning, followed by immunostaining. Asa result, many thereof were found to also be positive for NKX2-1 (FIG.8A). In addition, the cells induced to differentiate in the same mannerwith the use of SFTPC-reporter 201B7 were subjected to immunostaining.As a result, the presence of SFTPC- or proSPB-positive cells wasobserved in the CPM-positive cells (FIG. 8B). The percentage of theSFTPC-positive cells was found to be about 0.9% as a result of flowcytometric analysis (FIG. 8C).

Subsequently, the cells (201B7) after the completion of Step 5 (Day 25)were subjected to immunostaining, and CPM-, NKX2-1-, SFTPB-, SFTPC-, andCCSP-positive cells were identified (FIGS. 9A and 9B). In this case,marker genes were found to be also positive for CPM. In addition,CPM-positive cells obtained using MACS were subjected to quantitativeRT-PCR, so as to assay the CPM, NKX2-1, SFTPA2, SFTPB, DCLAMP, SFTPC,CCSP, and NGFR mRNA levels. As a result, sorting of CPM-positive cellswas found to result in a remarkable increase in the CPM and NKX2-1 mRNAlevels (FIG. 10).

As described above, it was demonstrated that alveolar epithelial cellsor progenitor cells thereof could be induced from iPS cells by themethod of the present invention.

[Effects of CPM Marker]

CPM expression in human fetus-derived lung tissue (FIG. 11) and mousefetus-derived lung tissue (E12.5, E15.5, and E17.5) (FIG. 12) wasinspected. As a result, expression of CPM in combination with NKX2-1,SFTPC, and T1α was observed. Accordingly, CPM was found to be capable ofrecognizing alveolar epithelial progenitor cells at an early stage, suchas during the prenatal period (humans: 3 to 7 weeks in the prenatalperiod; mice: 9 to 14 days in the prenatal period), in addition to thecanalicular period of lung development (humans: 16 to 24 weeks in theprenatal period; mice: 16.5 to 17.5 days in the prenatal period) and theadenoid period (humans: 7 to 16 weeks in the prenatal period; mice: 14.0to 16.5 days in the prenatal period).

As described above, it was found that alveolar epithelial progenitorcells could be recognized and extracted with the use of CPM as anindicator.

[Three-Dimensional Culture]

As described above, the CPM-positive cells (2×10⁴ cells) extracted viaMACS obtained after the completion of Step 3 with the use ofSFTPC-reporter 201B7 were transferred to 12-well Cell Culture Inserts(BD Biosciences) supplemented with 400 μ1 of medium containing Matrigelin combination with basal medium 3 containing 50 nM dexamethasone, 0.1mM 8-Br-cAMP, 0.1 mM IBMX, and 10 ng/ml KGF at a ratio of 1:1 togetherwith 2×10⁶ human fetus-derived pulmonary fibroblasts (PP002-F-1349, DVBiologics). Also, basal medium 3 containing 10 μM Y-27632, 50 nMdexamethasone, 0.1 mM 8-Br-cAMP, 0.1 mM IBMX, and 10 ng/ml KGF was addedto the lower layer of the Cell Culture Inserts so as to form spheroids(cell masses), and culture was conducted for 10 to 12 days (FIG. 13).The resulting spheroids were inspected using a transmission electronmicroscope and found to be cells having lamella-like structures (FIG.14A).

In addition, the spheroids were subjected to hematoxylin-eosin staining,and CPM(+)-cell-derived spheroids were found to be in the form of cysticpseudo-lamellar, cylindrical, or cubic cells having cytoplasm that wouldbe stained dark pink, unlike CPM(−)-cell-derived spheroids havingcytoplasm of pale color (FIG. 15A). These cells were double positive forboth NKX2-1 and CPM. In addition, these cells included SFTPC-positivecells (FIG. 15B). In this case, AQPS-positive cells as markers of type Ialveolar epithelial cells were found to be present adjacent toSFTPC-positive cells.

The alveolar epithelial cell marker expression was inspected and SFTPA,SFTPB, SFTPC, and SFTPD were found to be expressed in the CPM- andNKX2-1-positive cells (FIG. 16). As a result of quantitative PCR, theexpression levels of these genes were found to have been elevated viathree-dimensional culture. In addition, expression of SOX9 and ID2,which are indicators for induction of the peripheral airway, wasobserved in CPM- and NKX2-1-positive cells in several spheroids (FIG.16).

While PDPN and CAV1 were expressed in fibroblast-like cells in thevicinity of the spheroids (indicated with arrows), they were alsoexpressed in the spheroids (indicated with arrowheads) (FIG. 17).

As described above, it was found that alveolar epithelial cells wereinduced from CPM-positive cells, and CPM was found to be a useful markerof progenitor cells of alveolar epithelial cells. In addition, theCPM-positive cells obtained were found to be induced into maturealveolar epithelial cells via three-dimensional co-culture thereof withhuman fetus-derived pulmonary fibroblasts.

INDUSTRIAL APPLICABILITY

The method of the present invention enables production of alveolarepithelial progenitor cells from pluripotent stem cells.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. A cell population predominantly comprising carboxypeptidase M(CPM)-positive cells as alveolar epithelial progenitor cells or alveolarepithelial cells.
 2. The cell population according to claim 1, whereinthe cell population is produced by extracting CPM-positive cells, asalveolar epithelial progenitor cells, from a cell population comprisingalveolar epithelial progenitor cells using CPM as a marker, therebyobtaining a cell population predominantly comprising CPM-positive cells.3. A method for producing a cell population predominantly comprisingCPM-positive cells as alveolar epithelial progenitor cells comprisingextracting CPM-positive cells as alveolar epithelial progenitor cellsusing CPM as a marker, from a cell population comprising alveolarepithelial progenitor cells, thereby obtaining a cell populationpredominantly comprising CPM-positive cells.
 4. The cell populationaccording to claim 1, wherein the cell population is produced by amethod for producing alveolar epithelial progenitor cells frompluripotent stem cells comprising Steps (1) to (6): (1) culturingpluripotent stem cells in a medium containing activin A and a glycogensynthase kinase 3β (GSK3β) inhibitor; (2) culturing the cells obtainedin Step (1) in a medium containing a bone morphogenic protein (BMP)inhibitor and a transforming growth factor β (TGFβ) inhibitor; (3)culturing the cells obtained in Step (2) in a medium containing BMP4,retinoic acid, and a GSK3β inhibitor; (4) culturing the cells obtainedfrom Step (3) in a medium containing a fibroblast growth factor 10(FGF10); (5) culturing the cells obtained from Step (4) in a mediumcontaining a steroid drug, a cyclic adenosine monophosphate (cAMP)derivative, a phosphodiesterase inhibitor, and a keratinocyte growthfactor (KGF); and (6) extracting CPM-positive cells as alveolarepithelial progenitor cells using the CPM as a marker following Step(5); thereby obtaining a cell population predominantly comprisingCPM-positive cells.
 5. The cell population according to claim 4, whereinthe medium of Step (1) further comprises a Rho kinase (ROCK) inhibitorand/or a histone deacetylase (HDAC) inhibitor.
 6. The cell populationaccording to claim 4, wherein Step (1) comprises culturing for 6 days orlonger, Step (2) comprises culturing for 4 days or longer, Step (3)comprises culturing for 4 days or longer, Step (4) comprises culturingfor 7 days or longer and Step (5) comprises culturing for 4 days orlonger.
 7. The cell population according to claim 4, wherein the GSK3βinhibitor is CHIR99021, the BMP inhibitor is Noggin, the TGFβ inhibitoris SB431542, the steroid drug is dexamethasone, the cAMP derivative is8Br-cAMP, and the phosphodiesterase inhibitor is3-isobutyl-1-methylxanthine (IBMX).
 8. The cell population according toclaim 1, wherein the cell population is produced by a method forproducing alveolar epithelial progenitor cells from pluripotent stemcells comprising Steps (1) to (3b): (1) culturing pluripotent stem cellsin a medium containing activin A and a glycogen synthase kinase 3β(GSK3β) inhibitor; (2) culturing the cells obtained in Step (1) in amedium containing a bone morphogenic protein (BMP) inhibitor and atransforming growth factor β (TGFβ) inhibitor; (3a) culturing the cellsobtained in Step (2) in a medium containing BMP4, retinoic acid, and aGSK3β inhibitor; and (3b) extracting CPM-positive cells as alveolarepithelial progenitor cells using the CPM as a marker following Step(3a); thereby obtaining a cell population predominantly comprisingCPM-positive cells.
 9. The cell population according to claim 8, whereinthe medium of Step (1) in the method further comprises a Rho kinase(ROCK) inhibitor and/or a histone deacetylase (HDAC) inhibitor.
 10. Thecell population according to claim 8, wherein Step (1) comprisesculturing for 6 days or longer, Step (2) comprises culturing for 4 daysor longer, and Step (3a) comprises culturing for 4 days or longer. 11.The cell population according to claim 8, wherein the GSK3β inhibitor isCHIR99021, the BMP inhibitor is Noggin, and the TGFβ inhibitor isSB431542.
 12. The cell population according to claim 1, wherein the cellpopulation is produced by a method for producing alveolar epithelialcells from pluripotent stem cells comprising Steps (1) to (3c): (1)culturing pluripotent stem cells in a medium containing activin A and aglycogen synthase kinase 3β (GSK3β) inhibitor; (2) culturing the cellsobtained in Step (1) in a medium containing a bone morphogenic protein(BMP) inhibitor and a transforming growth factor β (TGFβ) inhibitor;(3a) culturing the cells obtained in Step (2) in a medium containingBMP4, retinoic acid, and a GSK3β inhibitor; (3b) extracting CPM-positivecells as alveolar epithelial progenitor cells using the CPM as a markerfollowing Step (3a); and (3c) co-culturing the extracted alveolarepithelial progenitor cells together with human fetal pulmonaryfibroblasts, following Step (3b); thereby obtaining a cell populationpredominantly comprising CPM-positive cells.
 13. The cell populationaccording to claim 12, wherein the medium of Step (1) further comprisesa Rho kinase (ROCK) inhibitor and/or a histone deacetylase (HDAC)inhibitor.
 14. The cell population according to claim 12, wherein Step(1) comprises culturing for 6 days or longer, Step (2) comprisesculturing for 4 days or longer, and Step (3a) comprises culturing for 4days or longer.
 15. The cell population according to claim 12, whereinthe GSK3β inhibitor is CHIR99021, the BMP inhibitor is Noggin, and theTGFβ inhibitor is SB431542.
 16. The cell population according to claim12, wherein step (3c) is conducted by co-culturing in three-dimensionalcell culture.
 17. The cell population according to claim 1, wherein theCPM-positive cells are human CPM-positive cells.
 18. The cell populationaccording to claim 1, wherein the proportion of CPM-positive cellsrelative to total cells in the cell population is at least 50%.
 19. Themethod according to claim 3, wherein the CPM-positive cells are humanCPM-positive cells.
 20. The method according to claim 3, wherein theproportion of CPM-positive cells relative to total cells in the obtainedcell population is at least 50%.
 21. An agent for treatment of apulmonary alveolar disease comprising the cell population according toclaim
 1. 22. A method for treating a pulmonary alveolar diseasecomprising a step of administering the cell population according toclaim 1 to a patient afflicted with a disease that destroy pulmonaryalveolus.
 23. The method according to claim 22, wherein a sheetcomprising the cell population is applied to alveolar epithelium of thepatient.
 24. The method according to claim 22, wherein a suspensioncomprising the cell population is implanted in pulmonary alveolus of thepatient.